AU565768B2 - Infectious bursal disease virus vaccine - Google Patents

Description

INFECTIOUS BURSAL DISEASE VIRUS VACCINE

This invention relates to the identification and characterisation of the major structural protein of infectious bursal disease (IBD) virus of chickens (host-protective immunogen) which stimulates the production of antibody that neutralises the infectivity of IBD virus in vitro and which protects susceptible chickens against infection with virulent IBD virus. The invention further relates to the production of an effective sub-unit vaccine against the virus utilising this major host-protective immunogen, as well as to the use of this immunogen in diagnostic tests, assays and the like.

Infectious bursal disease virus is a pathogen of major economic importance throughout the world poultry industry and is a ubiquitous contaminant of commercial poultry environments. The virus causes a highly contagious immunodepressive disease of young chickens, and selectively proliferates in the bursa of Fabricius (one of the two major avian immunological organs) thereby destroying the precursors of the antibody producing plasma cells. In young chickens (day old to 4 weeks) it directly causes morbidity and mortality, while the capacity to produce antibody responses is inhibited or depressed in chickens which survive infection. Such chickens respond poorly to vaccination programs aimed at other avian infections, remain highly susceptible to a variety of bacterial, mycoplasmal and viral pathogens and exhibit very poor weight gain and food conversion ratios.

Protection against IBD virus infection is mediated by humoral antibody alone and does not require the presence of cell-mediated immune effector systems. Thus chickens that receive an adequate amount of maternal antibody from an immune breeder hen via the yolk sac are protected through the critical first 4 to 5 weeks after hatching.

Current vaccination strategies are aimed at achieving the deposition of high levels of maternal antibody in fertilized eggs to protect the chickens throughout these critical weeks after hatching. The presently used vaccination regimens to control IBDV involve injecting breeder hens (previously exposed to live IBDV) with an inactivated oil-emulsion whole virus vaccine prior to the onset of their period of egg production around 22 weeks of age. This inactivated vaccine provokes a major secondary antibody response that is several orders of magnitude (> 100 fold) greater and persists for longer than the responses obtained by repeated vaccination with live virus. This results in the transmission of high levels of protective antibody to each egg throughout the next 40 weeks or so of the egg production cycle. If the antibody levels can be boosted sufficiently it should be possible for broiler chickens, that are slaughtered at 6 weeks of age, to go through their complete rearing period totally protected from IBD virus by maternally derived antibody.

The presently available inactivated IBD virus vaccine is expensive and difficult to produce since the virus cannot be grown to sufficiently high titres in simple culture systems such as embryonated eggs or tissue culture. Currently the viral material required for vaccine production is obtained by infecting six-week-old specified-pathogen-free (SPF) chickens and then harvesting the virus from the infected bursae 3 or 4 days after infection. This procedure for producing an IBD virus vaccine from infected SPF chickens is both laborious and expensive. The identification and isolation of the major host-protective immunogen in accordance with the present invention opens the way for development of a safe and inexpensive sub-unit vaccine which is effective in stimulating prolonged high-titre antibody responses in hens to allow the transfer of sufficient maternal antibody to protect young chickens, at least during the critical first few weeks after hatching.

One object of the work leading to the present invention has been to identify the IBD virus-encoded protein (s) that induce antibody in chickens following natural infection or the injection of a commercial inactivated oil-emulsion whole virus vaccine.

In this work, the purification of the

Australian strain of IBD virus from chicken bursae by successive rate-zonal and density-equilibrium centrifugation using sucrose and caesium chloride respectively has been studied. The virus so purified has been analysed and found to be composed of two major structural protein or polypeptide components of molecular weights (MW) approximately 37 kilodalton

(Kd) and 32 Kd, and three other components of MWs approximately 91.5 Kd, 41.5 Kd and 29 Kd. Only the major polypeptides reacted strongly with serum from chickens naturally infected or hyperimmunized with IBD virus, and of these the reaction of the 32 Kd polypeptide with antibody was typically the most intense. The 32 Kd polypeptide was a major component of all preparations (as revealed by Coomassie-blue staining of gels) of purified bursal grown Australian IBD virus, with a buoyant density in CsC1 of 1.33 g/ml. The relative amounts of the other polypeptides varied between preparations. It should be noted that from preliminary work in this regard, the 32 Kd polypeptide was estimated to have a molecular weight of approximately 31 Kd when compared with standard molecular weight markers. As a result of further work, however, the molecular weight of approximately 32 Kd is considered to be more accurate.

The major 32Kd polypeptide of the Australian isolate is comparable in size to the VP-3 protein (MW of 32 to 35 Kd) detected in studies overseas on the Cu-1 isolate of IBD virus grown in vitro or in vivo (Nick et al, 1976; Dobos 1979; Todd & McNulty, 1979; Muller & Becht, 1982). The major 37 Kd polypeptide is, however, smaller than the VP-2 (MW of 40 to 41 Kd) of overseas isolates, while the VP-X protein (MW of 47 to 48 Kd - Dobos, 1979; Muller & Becht 1982) was not detected in preparations of the intact Australian virus. The 41.5 Kd polypeptide of the Australian isolate is, however, the precursor of the 37 Kd polypeptide, and it is suggested that the 41.5 Kd polypeptide is analogous to VP-X of overseas isolates.

It has now been demonstrated that serum from naturally infected or hyperimmunised chickens contains antibodies to all polypeptides of the Australian isolate other than the minor 91.5 Kd polypeptide. It has also been shown that antibodies specific for the 32 Kd polypeptide of IBD virus appear first during the primary immune response of SPF chickens to the infectious virus, and these are also the predominant antibodies detected in the primary response of chickens to an inactivated whole virus vaccine prepared from IBD virus propogated in the bursa. Only later in the response to live virus or following revaccination with an inactivated vaccine could antibodies to other structural polypeptides be readily detected. The 32 Kd polypeptide thus appears to be a major, if not the major, immunogen of IBD virus.

The protective ability of chicken sera which contain only antibodies specific for the 32 Kd polypeptide, as assessed by Western blotting analysis on viral polypeptides separated by SDS-PAGE, is attested to by the capacity of such sera to neutralise the infectivity of IBD virus in vitro and to passively protect highly-susceptible 2-day-old SPF chickens. Furthermore, chickens immunised with the purified 32 Kd polypeptide produced antibody detectable by ELISA and the virus neutralisation assay, while chickens immunised with the 37 Kd or 41.5 Kd polypeptides produced antibody detectable by ELISA, but only low levels of virus neutralising ability. Adsorption of the anti-32 Kd sera with the 37 Kd or 41.5 Kd polypeptides did not reduce the virus neutralising titre of the anti-32 Kd sera. These results confirm that the 32 Kd polypeptide is a major protective immunogen of IBD virus.

According to one aspect of the present invention, there is provided a non-infectious sub-unit vaccine for use against IBD virus which comprises the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, together with, if desired, an adjuvant.

In a related aspect, this invention provides a method of increasing the level of protective antibodies against IBD virus in poultry, particularly breeding hens, which method comprises administering the aforesaid vaccine to said poultry.

In yet another aspect, this invention provides a method of providing passive immunity to IBD virus in poultry, which method comprises administering to said poultry an antiserum containing antibodies specific for the 32 Kd structural polypeptide or an immunogenic peptide derived therefrom.

The 32 Kd polypeptide may be isolated from

IBD virus , for example IBD virus which has been grown in and purified from infected bursae of Fabricius in chickens.

As mentioned above, the vaccine according to this invention may comprise an immunogenic peptide derived from the 32 Kd polypeptide, for example, by "genetic engineering" or chemical synthesis. A suitable immunogenic peptide may be derived so that it comprises all or at least the major immunogenic determinants of the 32 Kd polypeptide contained in the IBD virus and thus exhibits the same or similar immunogenicity to the 32 Kd polypeptide. If required the 32 Kd polypeptide may also be coupled to a carrier molecule to increase its immunogenicity and hence its efficacy as a vaccine.

Preferably, the non-infectious sub-unit vaccine of this invention comprises an adjuvant. The vaccine may, for example, be delivered in an aqueous-mineral oil emulsion, such as an emulsion achieved by using an oil-phase emulsifier (e.g. Arlacel 80) and an aqueous-phase emulsifier (e.g. Tween 80) as described by Stone et al., 1978.

Additional adjuvants may also be included if required, for example Al OH₃ (Wells et al . , 1979 ) , saponin or a derivative of muramyl dipeptide (Wells at al., 1982).

In other aspects of the present invention there are provided methods for assaying both quantitatively and qualitatively the levels of protective antibodies in poultry, including breeding hens and their progeny, and methods for assaying the relative concentrations of protective antigen in preparations of IBD virus produced for experimental and commercial inactivated vaccines, which methods are characterised by the use as an immunogen of the polypeptide of approximate MW 32 Kd isolated from IBD virus, or an immunogenic peptide derived therefrom. Further details of the methods by which these immunoassays can be carried out are well known in the art, and are accordingly not described in detail here. These methods include the well known ELISA and radioimmunoassays.

The following detailed description relates to the isolation and characterisation by electrophoresis and Western blotting of an Australian isolate of IBD virus. In the accompanying diagrams: Figure 1 shows the electrophoretic profile of the total RNA isolated from IBD virus which had been fractionated on a 25 to 50% sucrose gradient (10 ml) at 28,000 rpm for 90 min. The white doublets towards the top of the gel in fractions

3, 4 and 9 from the gradient comprise the two segments of ds-RNA present in IBD virus. Arrow indicates increasing concentration of sucrose.

Figure 2 shows (a) Polyacrylamide gel electrophoresis of purified IBD virus in a 12.5% gel using the discontinuous SDS-gel system described by Laemmli (1970) . The gel was stained with Coomassie brilliant blue to reveal 2 major bands of approximate MW 37 Kd and 32 Kd and 3 others (arrowed) of approximate MW 91.5 Kd, 41.5 Kd and 29 Kd.

(b) Autoradiograph of a Western blot of the virus preparation from (a) after probing with serum from a chicken experimentally infected with live IBD virus 2 months previously. Molecular weight standards are on the left side of each gel

[ (a) Pharmacia standards and (b) Amersham 1⁴C-standards].

Figure 3 shows the specificity of the serum antibody response of a 6 week-old chicken to live IBD virus, as assessed by reacting 1:500 dilutions of serum collected 3,5,7,10 and 14 days after infection with Western blots of the viral polypeptides separated by SDS-PAGE. 14C-MW markers (Amersham, U.K.) are on the left-hand side. Figure 4 shows (a) the specificity of antibodies in serum collected from six 6-week-old chickens, 14 days after they had been infected with IBD virus (tracks 1-6) or in the serum from a chicken that had been infected 28 days previously (track 7).

Only one (track 1) of the 6 chickens had antibody with demonstrate specificity for other than the 32 Kd structures polypeptide by 14 days after infection. Autoradiograph exposed for 7 days. Amersham 14C-MW markers on left-hand side.

Figure 5 shows the specificity of the primary antibody response of 2 SPF chickens (1 and 2) injected at 5 weeks of age with a commercial inactivated oil-emulsion vaccine. Serum obtained from the chickens 4 and 8 weeks (a and b) after primary vaccination are compared with serum obtained 4 weeks (c) after a second injection of inactivated vaccine at 13 weeks of age. Amersham

¹⁴C-MW markers on left-hand side.

Figure 6 shows the specificity of antibodies in sera from a chicken infected at 5 weeks of age with live virus when examined 4 and 20 weeks post- infection (a and b). The chicken was then reimmunised with a commercial inactivated whole-virus vaccine at 25 weeks of age and the specificity of the response examined 4 and 8 weeks later (c) and (d). Amersham 14C-MW markers on left-hand side. MATERIALS AND METHODS

Growth and purification of IBD virus.

The isolate 002/73 of IBD virus was originally obtained in Australia by Firth (1974) from commercial poultry with varying degrees of bursitis and identified serologically as IBD virus at the Central Veterinary Laboratory, Weybridge, UK. Following propagation at a limiting dilution of infectivity, the virus was routinely propagated by intraocular inoculation of 4 to 6 week old specified pathogen free (SPF) white leghorn chickens (CSIRO SPF Poultry Unit - Maribyrnong, Victoria, Aust.). Homogenates of infected bursae of Fabricius were prepared as 10% (w/v) suspensions in phosphate buffered saline (PBS) and stored at -80°C. The IBD virus stocks appeared to be free of contamination by other poultry viruses on electromicroscopic examination and did not cross-react in the agar-gel precipitation test with antisera to avian reovirus.

The virus was purified by a modification of the method of Todd & McNulty (1979). An equal weight of chilled PBS was added to the freshly harvested bursae which were homogenised in an ice bath by 3 x 20s bursts of a Polytron (PT-10-OD, Kinematica, GMBH, Luzern, Schweiz) on setting 5. The hσmogenate was frozen to -80°C and thawed rapidly before an equal volume of the fluorocarbon Arklone (Wertheim Labs . , Melb . Australia) was added and the mixture rehomogenised. After centrifugation at 10,000 g for 30 min at 5°C, the aqueous phase (ca 7ml) was prepared in 0.1 M NaCl, 0.01 M Tris-HCl buffer, pH 7.6. After centrifugation at 28,000 rpm for 1.5 h in a Beckmann SW28 rotor at 5°C the gradients were harvested from the bottom in 1 ml fractions. These were then examined by gel electrophoresis, Western blotting with hyperiramune sera, ELISA and by an assay for ds-RNA in order to determine the position of complete or incomplete particles of virus and soluble proteins of the virus (in later preparations the virus was collected from the interface of a stepwise gradient prepared by overlaying 10 ml of 40% sucrose on 5 ml of 60% sucrose). Fractions containing complete (intact) virus were pooled and layered onto chilled preformed 20 to 40% (w/v) CsCl gradients (10 ml) which were centrifuged at 30,000 rpm for 18 h at 5°C in a Beckmann SW40 Ti rotor and the band of IBD virus harvested through the side of the tube. The virus was dialysed against NaCl-Tris buffer to remove CsCl and then against NaCl-Tris buffer containing 0.05% (w/v) sodium azide before being stored at 4°C or else made 50% (v/v) in glycerol and stored at -20°C.

Chicken antisera to IBD virus

SPF chickens were infected intraocularly with IBD virus and bled 0,3,5,7,10 and 14 days later, then every two weeks. Chickens injected intramuscularly with 0.5 ml of commercial inactivated oil-emulsion vaccine (Arthur Webster Pty.Ltd., Northmead, NSW, Aust.) were bled fortnightly, as were chickens given a second intraocular infection with IBD virus or previously-infected chickens given an intramuscular injection of commercial inactivated vaccine. The sera were collected by centrifugation at 400 g for 15 min and stored at -20°C. Enzyme linked immunosorbent assay (ELISA)

The ELISA method used to assess the presence of IBD viral antigen in various gradient fractions or chicken antibody to IBD virus was essentially that described by York et al., (1983), except that the microtiter trays (Nunc Immunoplate I) were coated with rabbit anti-IBD virus IgG prepared by hyperimmunising rabbits with 002/73. To detect viral antigens in the gradient, serial dilutions of the fractions were added to the wells, which were then treated with a dilution of chicken anti-serum to IBD virus which produced a maximum OD_450nm of 1.0. To titrate antibodies to IBD virus, dilutions of chicken sera were added to the wells that had first been coated with rabbit antibody followed by a standardised concentration of an extract of infected bursae. The amount of chicken antibody binding to the viral antigen in each assay was then quantitated by adding sheep IgG-anti-chicken IgG-horseradish peroxidase conjugate, followed by

5-aminosalicylic acid. The trays were shaken for 30 min and the OD_450nm was then read immediately on a Titertek Multiscan (Flow Laboratories, Australia).

Polyacrylamide gel electrophoresis (PAGE) and Western blotting.

Aliquots (40μl) of each sucrose-gradient fraction were dried down under vacuum, resuspended in 20μl of the sample buffer described by Laemmli (1970) which contained SDS and a trace of solid bromophenol-blue dye, then heated for 3 min in a boiling water bath. These samples were examined by discontinuous PAGE (Laemmi, 1970) with Coomassie blue staining. The structural proteins of the virus, separated by SDS-PAGE, were also examined by the Western blotting procedure described by Burnette (1981). The viral proteins were transferred to nitrocellulose membrane-filter (Schleicher and Schύll BA83 0.2μm) and probed with chicken antisera diluted 1: 500 in 1% (w/v) gelatin in NaCl-Tris buffer. The chicken antibodies binding to viral polypeptides were identified with rabbit IgG anti-chicken IgG (Cappel Labs, Cochranville, USA) diluted 1:1000 in NaCl-Tris-gelatin buffer followed by IμCi of

125 I-Protein A (Amersham, U.K.) in the same buffer.

An autoradiograph of the nitrocellulose membrane was prepared using Fuji Rx Medical X-ray film and Ilford

Fast Tungstate intensifying screens usually for 16-24 h at -70°C.

Detection of IBD virus by the RNA content of various fractions.

Sucrose-gradient fractions were diluted to 1:4 with 10 mM Tris-HCl, 50 mM NaCl, 0.2% SDS buffer, pH 7.05 and treated with 0.5 mg/ml ribonuclease-free Pronase (Worthington, USA) for 1 h at 37°C. The solutions were made 0.3 M with respect to NaCl and the nucleic acids extracted with.1 volume of phenol at 56°C for 5 min. One volume of chloroform was added to the mixture which was then shaken at room temperature for 10 min before being centrifuged at 12,000 rpm for 2 min in an micro-centrifuge (Eppendorf, W.Germany). The nucleic acids in the upper aqueous phase were recovered by precipitation with 2 volumes of ethanol at -20 °C for 30 min followed by centrifugation. The RNA pellets were washed thoroughly with 67% (v/v) ethanol, dried and then dissolved in 20μl water. Samples of RNA were electrophoresed under non-denaturing conditions in 1% (w/v) agarose slab gels in 20 mM phosphate buffer, pH 6.8, together with

DNA standards (Boehringer, W.Germany). When the gels were stained with acridine orange and illuminated with UV light, the ds-DNA or RNA appeared as green bands while single-stranded (ss) RNA appeared as red bands (McMaster & Carmichael, 1977).

RESULTS

Purification of IBDV from infected bursae

Following centrifugation of the clarified bursal homogenates in 25 to 50% continuous sucrose gradients, a major band of material was visible approximately 3/5 the way down the gradient. SDS-PAGE analysis of the sucrose fractions indicated that the highest concentration of viral proteins was in the visible band, while fractions immediately above and below the major band contained lesser amounts of the viral proteins. Similarly, the ELISA for. viral antigen revealed peaks of IBD viral antigen throughout the gradient, although the visible band again contained the highest relative concentration.

The electrophoretic profiles of total RNA from different sucrose-gradient fractions located 2 segments of viral RNA in fractions 3 and 4, corresponding to the major visible band and in fraction 9 near the top of the gradient (Fig.l). Colour reaction with acridine orange showed that the two viral RNA segments were double-stranded. Fraction 10 contained only low MW RNAs (e.g. tRNA) and fractions 6-9 contained mainly ribosomal BNA (18S and 28S) and low MW RNAs. The viral ds-RNA bands were much more resistant to ribonuclease digestion than the ribosomal ss-RNAs. When electrophoresed under non-denaturing conditions with ds- DNA as standards, the two viral RNA segments appeared to have molecular weights of 2.5 x 10⁶ and 2.2 x 10⁶ respectively.

CsCl density-equilibrium centrifugation of complete virus from the continuous sucrose gradients revealed one major band which was visible under reflected light and had a mean buoyant density of 1.33 g/ml. When the crude virus from the interface of a 40-60% stepwise sucrose gradient was further purified on CsCl, a second, less dense, band was frequently seen which appeared by electron microscopy to contain a high proportion of "core" particles.

The effect of the duration of infection on the yield of virus

No band of IBD virus was visible in CsCl gradients of virus from bursae harvested from chickens 2 days after infection. A distinct band of virus was visible in CsCl gradients of bursae from chickens infected for 3 days, but was more diffuse when the virus was purified from bursae harvested 4 days after infection. An ELISA for IBD viral antigen also found maximum titres of antigen in bursal homogenates obtained 3 Or 4 days after infection. Consequently, virus was routinely prepared from bursae that were collected 3 days after infection.

Amino acid analysis of an acid hydrolysate, assuming a mean amino acid-residue weight of 110, showed that up to 250μg of viral protein could be obtained from a single bursa. Coomassie-blue staining of SDS-PAGE of purified virus

As shown in Fig.2, purified preparations of intact virus contained 2 major polypeptides with approximate MW of 37 Kd and 32 Kd, and 3 other components (arrows in Fig.2) with approximate MW of 91.5 Kd, 41.5 Kd and 29 Kd. Although the polypeptide of MW 32 Kd was a major component of all preparations of virus, densitometer tracings from polyacrylamide gels of different preparations of virus revealed that the relative amounts of the polypeptides varied between preparations.

The kinetics and specificity of the primary antibody response of chickens infected with IBD virus.

The appearance of serum antibody to IBD virus, as determined by ELISA, was followed in 6 SPF chickens infected intraocularly at 6 weeks of age with isolate 002/73. Antibody was first detected on day 5 (mean titre 250), rising quickly to a mean titre of 10,000 on day 10 and 17,000 on day 14. The analyses by Western blotting of the sera obtained from one of these chickens is shown in Fig.3. Antibody binding to the 32 Kd polypeptide of IBD virus was detected on day 5, with the intensity of binding increasing with time after infection. The antibodies present in the circulation of this chicken remained relatively specific for the 32 Kd polypeptide, at least until day 14 of the response. Western blots of the antibody present in sera obtained 14 days after infection from all 6 chickens are shown in Fig.4 (tracks 1-6). The sera from 5 of these 6 chickens demonstrated specificity for the 32 Kd polypeptide, even when the autoradiographs were exposed for 7 days. By 28 days after a primary infection the chickens had produced antibodies to all IBD virus polypeptides, except the 91.5 Kd polypeptide (Fig.4, track 7).

Response of SPF chickens to vaccination with an inactivated oil-emulsion IBD vaccine

Six chickens, when 5 weeks of age, were injected intramuscularly with 0.5 ml of a commercial inactivated whole virus vaccine. The sera from the 2 chickens with the highest ELISA titres at 8 weeks post-vaccination (titres of 25, 600 and 51, 200 respectively) were analysed by Western blotting. At 4 and 8 weeks after vaccination the primary antibody response of both chickens to the inactivated vaccine was relatively specific for the 32 Kd polypeptide of IBD virus (Fig.5, tracks a and b). By 4 weeks after a second intramuscular injection of inactivated vaccine at 13 weeks of age, however, both chickens had produced serum antibodies which reacted with the 3 most abundant structural proteins (Fig.5, tracks c) .

Response of sensitised chickens to an inactivated oil-emulsion IBD vaccine

Chickens that had been given live IBD virus at 5 weeks of age were injected at 25 weeks of age with a commercial inactivated vaccine. Sera were obtained 4 weeks and 20 weeks after the primary infection and then 4 and 8 weeks following revaccination. Analysis by Western blotting showed that initially there had been a response to 4 of the structural proteins, which had waned by 20 weeks post-infection (Fig.6). An injection of inactivated vaccine at that time resulted in a heightened response to all the IBD viral polypeptides, including the 91.5 Kd polypeptide (Fig.6, track d) .

The following detailed description describes experiments directed towards defining the major immunogen of IBD virus and assessing the protective efficacy of antibodies to that immunogen in vitro and in vivo. In the accompanying drawings:

Figure 7 shows Western-blots of immune serum collected from three 10-week-old chickens (tracks 1, 2 and 3), 14 days after infection with IBD virus (002/73). Nitrocellulose strips reacted with 25μl of serum diluted 1:100. Hyperimmune serum (track 4) included as a positive control.

Figure 8 shows (a) Protein profile (OD 280nm) of an S200 column fractionation of day 10 immune serum from a 10-week-old chicken infected with IBD virus (002/73).

(b) Antibody activity detectable by ELISA (OD 450nm) in a 1:10 dilution of each fraction.

Figure 9 shows Western-blots of whole virus with pools of the (a) IgM, and

(b) IgG fractions of 10 day immune sera obtained from two 10-week-old chickens (1 and 2) infected with IBD virus (002/73) .

Figure 10 shows Western-blots of sera from adult chickens obtained 3 weeks after immunisation with approximately 50μg of purified structural polypeptides of IBD virus (002/73). Chickens A, B and C immunised with 32 Kd polypeptide: D, E and F immunised with 37 Kd polypeptide: G, H and I immunised with 41.5 Kd polypeptide. Track K reacted with hyperimmune serum. Amersham C ¹⁴- MW markers on left-hand side.

Figure 11 shows Western-blots of 2 anti-32 Kd polypeptide sera (A and B) before (a) and after adsorption with (b) the 37 Kd polypeptide or (c) the 41.5 Kd polypeptide and of 2 anti-37 Kd polypeptide sera (E and F) before (a) and after adsorption with (d) the 32 Kd polypeptide. The extraneous antibody activity removed by adsorption is arrowed on each original serum (a) Refer to Table 5.

Figure 12 shows Western-blots of day 10 immune serum from a chicken infected with 002/73 (L) and day 28 immune serum from a chicken immunised with inactivated vaccine (K) before (a) and after adsorption with the (b) 32 Kd, (c) 37 Kd or (d) 41.5 Kd polypeptides. Refer to Table 5.

MATERIALS AND METHODS

Animals.

White Leghorn chickens of the "CSIRO-W" line were supplied by the CSIRO Specified Pathogen Free (SPF)

Poultry Unit, Maribyrnong, Victoria and transferred into flexible-film plastic poultry isolators, Dennett and Bagust (1979) at one-day-old.

Virus

The Australian isolate of IBD virus (002/73) used in these studies was originally described by Firth (1974) and identified serologically as IBD virus at the Central Veterinary Laboratory, Weybridge, U.K. Following one passage at limiting dilution of infectivity, the virus was routinely passaged by intraocular (i.o.) infection of 4 to 6 week old SPF chickens_* Virus infectivity was titrated by inoculating 3-day-old SPF chickens i.o. with 25μl of log_ln dilutions of a 10% (w/v) homogenate of infected bursae. The bursae were harvested from these chickens 72h later , homogenised and IBD viral antigen detected by an enzyme-linked immunosorbent assay (ELISA). The titre of virus was expressed as the reciprocal of the dilution of virus that infected 50% of the chickens inoculated (CID₅₀) .

IBD virus adapted to propagate in chick embryo fibroblast (CEF) culture was initially made available by A.Webster Pty.Ltd., Sydney, Australia. The virus has been designated TC-IBD virus (GT101) and was routinely passaged in CEF cultures. GT101 virus is neutralised in vitro by chicken antisera to type-1 IBD virus, but not type-2 IBD virus (unpublished data - Central Veterinary Laboratory, Weybridge, UK).

Purification of virus.

IBD virus (002/73) was grown in bursae then purified as described above. Briefly, a 50% homogenate of the infected bursae in 0.01M Tris-HCl, 0.15M NaCl, pH 7.6 (TBS) was frozen/thawed and homogenised with an equal volume of the fluorocarbon Arklone (Wertheim Labs., Melbourne, Australia). The clarified aqueous phase was centrifuged on stepwise gradients of 40% and 60% (w/v) sucrose. The sucrose interface was collected and centrifuged on preformed 25% to 50% (w/v) CsCl gradients. The purified intact virus banded at a density on CsCl of 1.33g/ml.

Polyacrylamide gel electrophoresis (PAGE) and immunoblotting of viral proteins.

Viral polypeptides were analysed in 12.5% (w/v) polyacrylamide slab gels using the discontinuous SDS gel system of Laemmli (1970), then transferred from the gel onto nitrocellulose paper by the Western blotting technique described by Burnette (1981) and reacted with chicken antibodies as described above. Briefly, the nitrocellulose membrane filter was blocked with a 5% (w/v) solution of dried skim milk powder (blotto) in TBS (Johnson, et al , 1984 ) , cut into 5mm strips then reacted with a 1:100 dilution of chick antisera in blotto, followed by a 1:1000 dilution of rabbit anti-chicken IgG (Cappel Labs.,

Cochranville, USA) in blotto and finally 1 μCi of

¹²⁵I-Protein A (Amersham, U.K.). Autoradiographs were prepared using Fugi Medical X-ray film and Ilford Fast

Tungstate intensifying screens.

Purification of the structural polypeptides of IBD virus.

Purified virus was boiled with SDS for 2 min in the absence of reducing agents and the peptides separated by SDS-PAGE. The gels were lightly stained with Coomassie-blue, destained and the 29 Kd, 32 Kd, 37 Kd, 41.5 Kd and 91.5 Kd bands of protein cut from the gels. The polypeptides were eluted from the gel strips into 0.05M Tris-acetic acid buffer (pH8.0) containing 0.1% (w/v) SDS at 50 volts for 40h. The eluted polypeptides were dialysed against multiple changes of distilled H₂O for 48h and the approximate concentration of the polypeptides assessed by SDS-PAGE against known concentrations of MW markers (Pharmacia, Sweden). The purity of the polypeptides was assessed by Western-blotting with hyperimmune chicken serum.

Chicken antisera to whole virus or the structural polypeptides of IBD virus

Six to ten-week-old SPF chickens were inoculated i.o. with infectious virus (002/73) and bled 10 and 14 days later. The serum was collected by centrifugation and stored at -20°C. SPF chickens were also injected intramuscularly (i.m.) with 0.5ml of a commercial inactivated IBD vaccine (Arthur Webster, Pty.Ltd.), and sera collected 28 days later, at the peak of the primary antibody response.

Adult SPF chickens were injected i.m. with approximately 50μg of either the 32 Kd, 37 Kd or 41.5 Kd polypeptides of IBD virus emulsified in 2 volumes of Freund's complete adjuvant (FCA) (Difco, U.S.A.). Three weeks later the chickens were reinjected i.m. with the same amount of the respective polypeptides in Freund's incomplete adjuvant (FIA)

(Difco). Approximately 40ml bleeds were collected at approximately weekly intervals into preservative free heparin (a final concentration of 10U/ml) and the plasma collected and frozen at -20°C. When the plasma was thawed, the fibrin clot was removed by centrifugation. Other chickens were immunised with the 29 Kd and 91.5 Kd polypeptides, except that the amounts of polypeptide injected were very much less and the antibody responses were correspondingly weak. ELISA for anti-IBD virus antibody and IBD viral antigen.

Anti-IBD virus antibody in chicken sera and IBD viral antigen in bursal homogenates were both quantitated using the ELISA as described above.

Plaque-reduction serum neutralising (SN) assay

Serial dilutions of heat inactivated (56°C/30 min) serum in biocarbonate buffered medium 199 (Gibco, USA) were added to an equal volume of TC-IBD virus, which had been diluted to approximately 500 plaque-forming units (pfu)/ml. Virus-serum mixtures were incubated at room temperature for 60 min with occasional shaking and then 0.1ml of each mixture inoculated into 3 secondary CEF cultures (35mm diam.plastic petri dishes, Kayline, Sth.Australia) to test for residual viral infectivity. The virus was allowed to adsorb to monolayers for 60 min at 37°C before each dish was overlayed with 2ml of 0.7% (w/v) agar (Baco-Difco, USA) in HEPES (0.015 M) buffered medium 199 containing 5% (w/v) calf serum. The cultures were incubated at 37°C for a further 6 days and stained by the addition of 0.15% (w/v) neutral red in a 1% agar overlay. The end-point of the neutralisation assay was the dilution of serum which caused a 50% reduction in the number of IBD virus plaques.

The Micro-SN assay

Serial dilutions of inactivated serum were prepared in a volume of 25μl of 199 containing 10% (w/v) TPB (Difco) and 2% heat inactivated fetal calf serum in flat-bottomed microtiter trays (Linbro, USA) before an equal volume of TC-IBD virus (500 pfu/ml) was added and incubated at 37°C for lh. Following incubation, 50μl of a 7.5 x 10⁵ cell/ml suspension of secondary CEF cells were added and the trays incubated at 37°C for 5 days. The trays were then stained with 1% (w/v) crystal violet in methanol and the end point read as the last dilution to completely inhibit virus replication.

Passive protection of chickens with specific antibody

Chickens were injected intraperitoneally (i.p.) at 2 days of age with various immune serum or control serum free of antibody to IBD virus. One day later the chickens were challenged i.o. with 25μl of bursal homogenate, usually containing a minimum of 1000 CID50 of IBD virus 002/73. Three days after infection, the chickens were exsanguinated and their bursae removed, weighed and made into a 5% homogenate in saline. Both the levels of antibody in the sera and the presence of viral antigen in the bursae were quantitated by ELISA.

Column chromatography

Five ml of immune serum was separated on a 2.5cm x 100cm S200 (Pharmacia) column by elution with 0.01M phosphate, 0.15M NaCl, pH 7.6 (PBS). The fractions were assayed for antibody activity by ELISA before the IgM region and IgG regions were pooled, deleting one 6ml fraction overlapping the two regions. The pools were concentrated to 4ml using an XM100A membrane (Amicon, USA) and sterile filtered. Affinity Chromatography

Only the 32 Kd, 37 Kd and 41.5 Kd polypeptides were obtained in sufficient quantity to prepare absorption columns. The polypeptides were obtained from unstained gels, guided by Coomassie stained strips taken from each margin of the gel. The purity of the eluted polypeptides was assessed by Western blotting with hyperimmune serum. The polypeptides were quantitated by a Lowry protein assay (Hartree, 1972) and between 150 and 300 μg of the respective polypeptides were reacted with 1ml of Affigel 10 (Biorad, USA) according to the manufacturers instructions.

One ml of each antipolypeptide sera was run slowly through the adsorption columns and eluted with 3 volumes of PBS. The columns were reactivated by passing 5ml of 1M proprionic acid through the columns followed by 20ml of PBS. Sera from chickens immunised with whole virus or inactivated vaccine were adsorbed by running 0.25ml of serum onto the columns and eluting with 1.75ml of PBS. The effectiveness of the adsorption was assessed by immunoblotting.

RESULTS

Passive protection with monospecific anti-32 Kd sera

When antisera obtained from 6-week-old chickens, 14 days after the had been infected with IBD virus, were analysed by Western-blotting, 5 were found to contain only antibodies specific for the 32 Kd structural polypeptide. The sera had ELISA titers between 6,620 and 51,200 and virus neutralisation titers of 6,250 or greater (Table 1) . The ability of these sera to passively protect chickens was assessed by injecting lml of each serum into each of four 2-day-old chickens, which were challenged 1 day later with 1000 CID₅₀ of 002/73. All the chickens that received immune serum resisted infection and had circulating antibody when exsanguinated 3 days after challenge, while those that received antibody negative serum had no antibody and were susceptible to infection (Table 1) .

The experiment was repeated with day 10 and day 14 immune serum from three 10-week-old chickens infected with IBD virus. Again these sera were monospecific by Western-blotting (Fig.7) and had ELISA titers between 57,000 and 77,000 at day 10 and 70,000 and 166,000 at day 14. Two ml of each serum was injected into each of 3 chickens. In addition to the groups of chickens that were challenged with 1000 CID₅₀ of 002/73, small groups of chickens that received either pooled immune serum or control serum remained unchallenged and served as in-contact controls. Neither the chickens that received immune serum and virus (Table 2) nor the in-contact chickens were infected. The chickens that were given normal chicken serum and virus were all susceptible to infection.

Characterisation of the protective antibodies in anti-32 Kd serum

When sera from two 10-week-old chickens, obtained 10 days after infection with IBD virus, were fractionated by S200 column chromatography and assayed for antibody detectable by the ELISA, all activity was confined to the IgG region of the column profile (Fig.8). The IgM region and the IgG regions were pooled and titrated by the micro-SN assay. Both the IgM and IgG pools of these sera were found to have virus neutralising activity; the IgG pool having twice the SN-titer of the IgM pool (Table 3) . Immunoblotting whole virus with these antibody pools showed that this assay also primarily detected IgG antibodies which were specific for the 32 Kd polypeptide (Fig.9).

One ml of the IgM and IgG pools from each serum were injected into groups of four 2-day-old chickens, which were then challenged 1 day later with 10 CID₅₀ of virus. The IgG antibody pools were found to confer protection while chickens injected with the IgM pools of antibody were all susceptible to infection (Table 3).

Antibody responses to purified viral polypeptides

Adult SPF chickens did not produce substantial titers of antibody that could be detected by either the ELISA or micro-SN assay during the early stages of the response to approximately 50μg of either the 32 Kd, 37 Kd or 41.5 Kd purified viral polypeptides in FCA. Immunoblotting with serum obtained 3 weeks after immunisation, however, showed that the chickens had synthesised antibodies to their respective polypeptides (Fig.10). Chicken B, which produced the strongest response to the 32 Kd polypeptide, also produced antibodies to the 37 Kd and 41.5 Kd polypeptides (Fig.10, track B) and the sera from the 3 chickens (D, E and F) injected with the 37 Kd polypeptide were almost indistinguishable from sera from the 3 chickens (G, H and I) injected with the 41.5 Kd polypeptide (Fig.10, tracks D to I) . It was noted that all of the chickens (D to I) injected with either the 37 Kd or 41.5 Kd polypeptides also produced antibodies that reacted with low MW material on the blots, material that was not recognised by hyperimmune serum from vaccinated chickens (Fig.10, track K).

One week after a second injection of viral polypeptides in FIA, 1 of the 3 chickens injected with the 32 Kd polypeptide had a micro-SN titre of 256 (Table 4). Further bleedings at 3, 4 and 6 weeks after the second immunisation showed that 2 of the chickens injected with the 32 Kd polypeptide had neutralising titers for IBD virus of between 160 and 1280, while those injected with the 37 Kd or 41.5 Kd polypeptides had neutralising titers of 40 or less. One of the chickens injected with the 41.5 Kd polypeptides had the highest ELISA titers followed the second injection of polypeptides (Table 4) .

Affinity chromatography of anti-polypeptide sera

Probing Western blots of the virus with sera obtained 3 to 6 weeks after the second injection of the purified polypeptides showed that they all contained antibodies to the other polypeptides. Monospecific antisera were prepared by passing antipolypeptide sera through adsorption columns prepared by binding the various viral polypeptides to Affigel-10.

Passing the anti-32 Kd sera through a 37 Kd column removed all antibody a.ctivity to the 37 Kd and 41.5 Kd polypeptides (Fig.11, A. and B), but did not reduce the micro-SN titre (Table 5). The 41.5 Kd column was less efficient, but gave similar results in that it did not reduce the SN-titer of the anti-32 Kd sera. Passing the 2 anti-37 Kd sera with the highest SN activity, through the 32 Kd adsorption column removed all anti-32 Kd antibody (Fig.11, E and F) , but did not reduce the low levels of SN antibodies present in these sera (Table 5) . The more discriminating plaque-reduction assay confirmed that passing the anti-32 Kd sera through the 37 Kd column, did not reduce the activity in the sera (Table 6) . Passing the antipolypeptide sera through the homologous adsorption column either had no detectable effect on the micro-SN titre or reduced it by no more than 50% (Table 5) .

Day 10 serum from an infected chicken and day 28 serum from a chicken injected with an inactivated IBD vaccine, were also passed through the absorption columns. In neither case did the 37 Kd and 41.5 Kd columns affect the Western-blotting patterns (Fig.12) or the micro-SN titers of the serum (Table 5) . Passing the serum through the 32 Kd column, however, markedly reduced the intensity of the Western-blotting pattern (Fig.12) and reduced the micro-SN titre of the sera by half (Table 5) . In an attempt to improve the efficacy of absorption, day 10 serum was diluted 1:100 prior to being passed through the 32 Kd column. In this case, the plaque-reduction assay (Table 6) showed that the virus neutralising activity in a 1:20,000 dilution of serum was reduced by almost 50%.

Synergism between anti-polypeptide sera

Mixing equal volumes of an anti-32 Kd serum, which did not have a detectable micro-SN titre, with 3 different anti-37 Kd sera, did not enhance the SN titre of the mixtures more than could be accounted for by the activity of the anti-37 Kd sera alone (Table 7) . Similarly, mixing an anti-32 Kd serum which had a micro-SN titre of 320 with the 3 anti-37 Kd sera, resulted in mixtures with SN titers wich could be completely accounted for by the activity of the anti-32 Kd serum (Table 7).

Passive protection with anti-32Kd polypeptide serum

The concentration of antibody in 1 to 2ml of anti-32Kd polypeptide serum was found to be too low to produce detectable levels of circulating antibody when injected i.p. into young chickens. An (NH₄) ₂SO₄ precipitate of 20ml of anti-32Kd polypeptide serum (Chicken B) was prepared, redissolved in 4ml of PBS, sterile filtered and 1ml injected each of 3 chickens of 2 days of age. These chickens, together with 3 control chickens, were challenged one day later with 10 CID₅₀ of 002/73, exsanguinated 3 days after challenge and their bursae and serum assessed by the ELISA. The 3 chickens that received the precipitated antibody had residual ELISA titres between 160 and 320 and 2 of them had no detectable viral antigen in their bursae. The 3 control chickens had no detectable antibody and all had ELISA titres of, viral antigen >128.

Neutralisation of type-1 IBD viruses by chicken anti-serum specific for the 32Kd polypeptide.

Five SPF chickens of 7 weeks of age were infected with IBD virus (002/73) and bled on days 10, 11 and 12 post-infection. The 15 individual sera were assessed by Western-blotting, and all contained antibodies specific for the 32Kd polypeptide of IBD virus. The sera were pooled and sent to the Central Veterinary Laboratory, Weybridge, UK. When assessed by the serum neutralisation assay, the pooled serum was found to neutralise the type-1 strains PGB-98 , Cu-1 and G-13 in addition to GT-101, but not the type-2 IBD virus strain Ty-89.

REFERENCES

Burnette, W.N. 1981. "Western Blotting" : electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Analytical Biochemistry, 112 : 195-203.

Dennett, D.P. and Bagust, T.J. 1979. Application of a flexible-film isolator for rearing specific pathogen-free chickens and investigating poultry pathogens. Avian Pathology, 8 : 289-300.

Dobos, P. 1979. Peptide map comparison of the proteins of infectious bursal disease virus. J.Virol., 32 : 1046-1050.

Firth, G.A. 1974. Occurrence of an infectious bursal syndrome within an Australian poultry flock. Aust.Vet.J., 50 : 128-130.

Hartree, E.F. 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analytical Biochem. 48 : 422-427.

Johnson, D.A., Gautsσh, J.W., Sportsman, J.R., and Elder, J.H. 1984. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal.Techn., 1 : 3-8.

Laemmli, U.K. 1970. Clevage of structural proteins during assembly of the head of the bacteriophage T₄. Nature (Lond.), 227 : 680-685. McMaster, G.K., and Carmichael, G.G. 1977. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and ocridine orange. Proc.Natt.Acad.Sci., U.S.A., 74 : 4835-4838.

Muller, H. and Becht, H. 1982. Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements of infectious virus and incomplete particles. Journal of Virology, 44 : 384-392.

Nick, H., Cursiefen, D. and Becht, H. 1976. Structural and growth characteristics of infectious bursal disease virus. J.Virol., 18 : 227-234.

Stone, H.D., Brugh, M., Hopkins, S.R., Yoder, H.W., and Beard, C.W. 1978. Preparation of inactivated oil-emulsion vaccines with avian viral or mycόplasma antigens. Avian Diseases, 22 : 666.

Todd, D. and McNulty, M.S. 1979. Biochemical studies with infectious bursal disease virus: comparison of some of its properties with infectious pancreatic necrosis virus. Archives Virol., 60 : 265-277.

Wells, P.W., Gilmour, N.J.L., Burrells, C. and Thompson, D.A. 1979. A serological comparison of

Pasteurella haemolytica vaccines containing different adjuvants. Res.Vet.Sci., 27 : 248-250.

Wells, P.W., Emery, D.L., Hinson, C.A., Morrison, W.I. and Murray, M. 1982. Immunisation of cattle with a variant-specific surface antigen of

Trypanosoma brucei : influence of different adjuvants.

Infect. Immunity, 36 , 1-10.

York, J.J., Fahey, K.J. and Bagust, T.J. 1983. Development and evaluation of an ELISA for the detection of antibody to infectious laryngotracheitis virus in chickens. Avian Diseases, 27 : 409-421.

It will be appreciated that many modifications and variations may be made to the particular methods described above by way of illustration of the present invention, and that the present invention includes all such modifications which fall within the scope of the invention as broadly described above.

Claims (10)

1. A non-infectious sub-unit vaccine for use against IBD virus which comprises the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, together with, if desired, an adjuvant.

2. A vaccine according to claim 1, wherein said 32 Kd polypeptide is isolated from IBD virus.

3. A vaccine according to claim 1, comprising an immunogenic peptide comprising all or at least the major immunogenic determinants of said 32 Kd polypeptide.

4. A vaccine according to claim 1, wherein said 32 Kd polypeptide or said immunogenic peptide is coupled to a carrier molecule to increase its immunogenicity.

5. A vaccine according to any one of claims 1 to 4, wherein said adjuvant is an aqueous-mineral oil emulsion.

6. A method of increasing the level of protective antibodies in poultry, which method comprises administering a vaccine according to any one of claims 1 to 5 to said poultry in ovo or at any time after hatching.

7. A method according to claim 6, wherein said vaccine is administered to breeding hens prior to the onset of their period of egg production in order to protect the progeny of said breeding hens.

8. A method of providing passive immunity to IBD virus in poultry, which method comprises administering to said poultry an antiserum containing antibodies specific for the structural peptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom.

9. A method for assaying the levels of protective antibodies against IBD virus in poultry, characterised in that the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, is used as an immunogen in the said assay.

10. A method for assaying the levels of protective immunogen in preparations of IBD virus produced for use as vaccines, characterised in that the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, is used as an immunogen in the said assay.